bhf-ucl amendt-1998-1 The molecular basis of Rieger syndrome. Analysis of Pitx2 homeodomain protein activities. in vitro In vitro emsa electrophoretic mobility shift assay Gel retardation assay band shift band shift Gel retardation assay emsa autoradiography autoradiography Amendt B.A., Sutherland L.B., Semina E.V., Russo A.F. Cardiac - Interactions involving cardiac related proteins Pitx2 has been entered as being of Human origin based on other work by the first author -see pmid 10490637. brad-amendt@uiowa.edu J. Biol. Chem. (0021-9258) 1998 Only protein-protein interactions imex curation amendt-1998-2 The molecular basis of Rieger syndrome. Analysis of Pitx2 homeodomain protein activities. in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Amendt B.A., Sutherland L.B., Semina E.V., Russo A.F. Cardiac - Interactions involving cardiac related proteins Pitx2 has been entered as being of Human origin based on other work by the first author -see pmid 10490637. brad-amendt@uiowa.edu J. Biol. Chem. (0021-9258) 1998 Only protein-protein interactions imex curation q99697-3 Pituitary homeobox 2 ARP1A PITX2 ARP1 RGS RIEG RIEG1 ALL1-responsive protein ARP1 Homeobox protein PITX2 Paired-like homeodomain transcription factor 2 RIEG bicoid-related homeobox transcription factor Solurshin protein protein human Homo sapiens Human METNCRKLVSACVQLEKDKSQQGKNEDVGAEDPSKKKRQRRQRTHFTSQQLQELEATFQRNRYPDMSTREEIAVWTNLTEARVRVWFKNRRAKWRKRERNQQAELCKNGFGPQFNGLMQPYDDMYPGYSYNNWAAKGLTSASLSTKSFPFFNSMNVNPLSSQSMFSPPNSISSMSMSSSMVPSAVTGVPGSSLNSLNNLNNLSSPSLNSAVPTPACPYAPPTPPYVYRDTCNSSLASLRLKAKQHSSFGYASVQNPASNLSACQYAVDRPV Variant in position: 58:F->L (in ASGD4). bcd_probe Drosophila bicoid site oligonucleotide ss dna single stranded deoxyribonucleic acid ss DNA ss dna synthetic construct synthetic construct GATCCGCACGGCCCATCTAATCCCGTGGGATC pit-1 Pit-1 DNA binding element ss dna single stranded deoxyribonucleic acid ss DNA ss dna synthetic construct synthetic construct GATCCTGTCTTCCTGAATATGAATAAGAAATAAGGATC mu 9 Mu 9 ss dna single stranded deoxyribonucleic acid ss DNA ss dna synthetic construct synthetic construct GATCCGCACGGCCCAGTTAATATCGTGGGATC prd Prd ss dna single stranded deoxyribonucleic acid ss DNA ss dna synthetic construct synthetic construct GATCCGCACGGCCCATCTTTGACGTCGTGGGATC ftz Ftz ss dna single stranded deoxyribonucleic acid ss DNA ss dna synthetic construct synthetic construct GATCCGCACGGCCCAGTTAATGGCGTGGGATC nkx-2.1 Nkx-2.1 ss dna single stranded deoxyribonucleic acid ss DNA ss dna synthetic construct synthetic construct GATCCCACTGCCCAGTCAAGTGTTCGGATC pit1_rat Pituitary-specific positive transcription factor 1 Pou1f1 Ghf-1 Pit-1 Pit1 Growth hormone factor 1 protein protein rat Rattus norvegicus (Rat) Rat MSCQPFTSADTFIPLNSDASAALPLRMHHSAAEGLPASNHATNVMSTATGLHYSVPSCHYGNQPSTYGVMAGTLTPCLYKFPDHTLSHGFPPLHQPLLAEDPTASEFKQELRRKSKLVEEPIDMDSPEIRELEQFANEFKVRRIKLGYTQTNVGEALAAVHGSEFSQTTICRFENLQLSFKNACKLKAILSKWLEEAEQVGALYNEKVGANERKRKRRTTISIAAKDALERHFGEHSKPSSQEIMRMAEELNLEKEVVRVWFCNRRQREKRVKTSLNQSLFSISKEHLECR bcd-pitx2-2 1572356 n/a 1572358 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) n/a 1572359 unspecified role unspecified role bait bait region 32p radiolabel 32p radiolabel P32 32P 32P P32 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false synthetic construct synthetic construct direct interaction direct interaction false false false 1572356 Fig. 1C, 2A, 2C, 6 Pit-1 (P10037) bcd-pitx2-3 1572356 n/a 1572358 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) n/a 1572359 unspecified role unspecified role bait bait region 32p radiolabel 32p radiolabel P32 32P 32P P32 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false synthetic construct synthetic construct n/a 1572360 competitor competitor competitor unspecified role unspecified role synthetic construct synthetic construct physical association physical association false false false Fig. 2A, 2B Unlabeled Pit-1 DNA binding element caused 34% inhibition. bcd-pitx2-5 1572356 n/a 1572358 unspecified role unspecified role prey prey Q99697-3:p.Thr68Pro mutation mutation certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) n/a 1572361 competitor competitor competitor unspecified role unspecified role synthetic construct synthetic construct n/a 1572359 unspecified role unspecified role bait bait region 32p radiolabel 32p radiolabel P32 32P 32P P32 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false synthetic construct synthetic construct physical association physical association false false false Fig. 3A, 3B The mutant Pitx2 T68P protein showed an overall increase in binding to all of the competitor DNAs (except the unlabeled bicoid DNA). The ability of these nonbicoid DNA competitors to bind Pitx2 T68P demonstrates a slight loss of binding specificity by this mutant. bcd-pitx2-6 1572356 n/a 1572358 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) n/a 1572359 competitor competitor competitor unspecified role unspecified role synthetic construct synthetic construct n/a 1572359 unspecified role unspecified role bait bait region 32p radiolabel 32p radiolabel P32 32P 32P P32 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false synthetic construct synthetic construct physical association physical association false false false Fig. 2A, 2B Unlabeled bicoid DNA (Bcd) caused more than 85% inhibition. bcd-pitx2-7 1572356 n/a 1572358 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) n/a 1572362 competitor competitor competitor unspecified role unspecified role synthetic construct synthetic construct n/a 1572359 unspecified role unspecified role bait bait region 32p radiolabel 32p radiolabel P32 32P 32P P32 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false synthetic construct synthetic construct physical association physical association false false false Fig. 2A, 2B Unlabeled Prd caused less than 20% inhibition. bcd-pitx2-8 1572356 n/a 1572358 unspecified role unspecified role prey prey Q99697-3:p.Thr68Pro mutation mutation certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) n/a 1572359 competitor competitor competitor unspecified role unspecified role synthetic construct synthetic construct n/a 1572359 unspecified role unspecified role bait bait region 32p radiolabel 32p radiolabel P32 32P 32P P32 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false synthetic construct synthetic construct physical association physical association false false false Fig. 3A, 3B The mutant Pitx2 T68P protein showed an overall increase in binding to all of the competitor DNAs (except the unlabeled bicoid DNA). The ability of these nonbicoid DNA competitors to bind Pitx2 T68P demonstrates a slight loss of binding specificity by this mutant. bcd-pitx2-9 1572356 n/a 1572358 unspecified role unspecified role prey prey Q99697-3:p.Thr68Pro mutation mutation certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) n/a 1572362 competitor competitor competitor unspecified role unspecified role synthetic construct synthetic construct n/a 1572359 unspecified role unspecified role bait bait region 32p radiolabel 32p radiolabel P32 32P 32P P32 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false synthetic construct synthetic construct physical association physical association false false false Fig. 3A, 3B The mutant Pitx2 T68P protein showed an overall increase in binding to all of the competitor DNAs (except the unlabeled bicoid DNA). The ability of these nonbicoid DNA competitors to bind Pitx2 T68P demonstrates a slight loss of binding specificity by this mutant. bcd-pitx2-10 1572356 n/a 1572358 unspecified role unspecified role prey prey Q99697-3:p.Thr68Pro mutation mutation certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) n/a 1572359 unspecified role unspecified role bait bait region 32p radiolabel 32p radiolabel P32 32P 32P P32 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false synthetic construct synthetic construct n/a 1572360 competitor competitor competitor unspecified role unspecified role synthetic construct synthetic construct physical association physical association false false false Fig. 3A, 3B The mutant Pitx2 T68P protein showed an overall increase in binding to all of the competitor DNAs (except the unlabeled bicoid DNA). The ability of these nonbicoid DNA competitors to bind Pitx2 T68P demonstrates a slight loss of binding specificity by this mutant. bcd-pitx2-11 1572356 n/a 1572358 unspecified role unspecified role prey prey Q99697-3:p.Thr68Pro mutation mutation certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) n/a 1572363 competitor competitor competitor unspecified role unspecified role synthetic construct synthetic construct n/a 1572359 unspecified role unspecified role bait bait region 32p radiolabel 32p radiolabel P32 32P 32P P32 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false synthetic construct synthetic construct physical association physical association false false false Fig. 3A, 3B The mutant Pitx2 T68P protein showed an overall increase in binding to all of the competitor DNAs (except the unlabeled bicoid DNA). The ability of these nonbicoid DNA competitors to bind Pitx2 T68P demonstrates a slight loss of binding specificity by this mutant. bcd-pitx2-13 1572356 n/a 1572358 unspecified role unspecified role prey prey Q99697-3:p.Thr68Pro mutation mutation certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) n/a 1572359 unspecified role unspecified role bait bait region 32p radiolabel 32p radiolabel P32 32P 32P P32 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false synthetic construct synthetic construct n/a 1572364 competitor competitor competitor unspecified role unspecified role synthetic construct synthetic construct physical association physical association false false false Fig. 3A, 3B The mutant Pitx2 T68P protein showed an overall increase in binding to all of the competitor DNAs (except the unlabeled bicoid DNA). The ability of these nonbicoid DNA competitors to bind Pitx2 T68P demonstrates a slight loss of binding specificity by this mutant. bcd-pitx2-14 1572356 n/a 1572358 unspecified role unspecified role prey prey Q99697-3:p.Thr68Pro mutation mutation certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) n/a 1572359 unspecified role unspecified role bait bait region 32p radiolabel 32p radiolabel P32 32P 32P P32 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false synthetic construct synthetic construct direct interaction direct interaction false false false 1572356 Fig. 1C, 3A, 3C, 6 Pit-1 (P10037) EMSAs demonstrated binding of Pitx2 and Pitx2 T68P mutant to the bicoid element (Fig. 1C). Binding to Pitx2 L54Q mutant was not detected most likely because of protein instability. bcd-pitx2-17 1572356 n/a 1572358 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) n/a 1572365 stimulator stimulator unspecified role unspecified role Stoichiometry: 2.0 n/a 1572359 unspecified role unspecified role bait bait region 32p radiolabel 32p radiolabel P32 32P 32P P32 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false synthetic construct synthetic construct physical association physical association false false false Fig. 6 Pit-1 increased the amount of Pitx2-DNA bound complex approximately 2–3-fold. The Pit-1-enhanced binding of Pitx2 was competed by unlabeled bicoid DNA bcd-pitx2-18 1572356 n/a 1572365 stimulator stimulator unspecified role unspecified role Stoichiometry: 2.0 n/a 1572358 unspecified role unspecified role prey prey Q99697-3:p.Thr68Pro mutation mutation certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) n/a 1572359 unspecified role unspecified role bait bait region 32p radiolabel 32p radiolabel P32 32P 32P P32 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false synthetic construct synthetic construct physical association physical association false false false Fig. 6 Pit-1 had a 2-3 fold stimulatory effect on the binding of the mutant Pitx2 T68P to the bicoid probe. pitx2-pou1f1-2 1572357 n/a 1572358 unspecified role unspecified role bait bait n-terminus gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false ecoli Escherichia coli (strain K12) n/a 1572365 unspecified role unspecified role prey prey direct interaction direct interaction false false false Fig. 7 O93385-2 and P10037 increase expression at RNA level of ENSRNOG00000017374.